Immune checkpoints and ncRNAs: pioneering immunotherapy approaches for hematological malignancies

Abstract

Hematological malignancies are typically treated with chemotherapy and radiotherapy as the first-line conventional therapies. However, non-coding RNAs (ncRNAs) are a rapidly expanding field of study in cancer biology that influences the growth, differentiation, and proliferation of tumors by targeting immunological checkpoints. This study reviews the results of studies (from 2012 to 2024) that consider the immune checkpoints and ncRNAs in relation to hematological malignancies receiving immunotherapy. This article provides a summary of the latest advancements in immunotherapy for treating hematological malignancies, focusing on the role of immune checkpoints and ncRNAs in the immune response and their capacity for innovative strategies. The paper also discusses the function of immune checkpoints in maintaining immune homeostasis and how their dysregulation can contribute to developing leukemia and lymphoma. Finally, this research concludes with a discussion on the obstacles and future directions in this rapidly evolving field, emphasizing the need for continued research to fully harness the capacity of immune checkpoints and ncRNAs in immunotherapy for hematological malignancies.

Introduction

Anti-programmed death-1 (PD-1) and anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) immune checkpoint inhibition have drastically changed the solid tumor cancer therapy landscape [1, 2]. However, their efficacy in hematological malignancies has been less pronounced. It is recognized that cancerous cells can circumvent immune surveillance by activating immune checkpoints, which serve as inhibitors of the immune system. Immune checkpoint inhibitors (ICIs) have revolutionized solid tumor treatment by reactivating the anti-tumor effect mediated by T-lymphocytes [3–5]. A number of T cell immune checkpoint molecules, such as CTLA-4 and PD-1, have been shown to be viable targets for therapy because of their non-redundant control over T cell responses in malignancies that are hematological in nature [6]. Hematological malignancies have responded better to immunotherapeutic approaches such as stem cell transplantation and anti-cancer monoclonal antibodies; nevertheless, the clinical advantages of immune checkpoint blockade (ICB) are restricted to tumor forms with significant levels of immune cell infiltration [7, 8]. Long-term disease management is a common challenge for ICB therapy, underscoring the necessity for a thorough understanding of immunological microenvironments unique to each illness in order to improve effectiveness [9–11].

Non-coding RNAs (ncRNAs) play a pivotal role in leukemia therapy, especially in relation to immune checkpoints. ncRNAs are implicated in the primary mechanisms underlying the development of drug resistance [12, 13]. They regulate physiological and pathological processes. In cases of chemo-resistant leukemia, ncRNAs are known to modulate the onset and progression of drug resistance [12, 14].

According to recent studies, leukemia and other malignancies are closely associated with the dysregulated expression of non-coding RNA genes and their mutational range [15, 16]. Thus, ncRNAs could serve as therapeutic targets and be used as biomarkers for diagnosis or prognosis [17]. ncRNAs may change how immune cells and cancer stem cells interact with the tumor microenvironment, which might lead to immunotherapy resistance [18, 19]. Immunologically associated ncRNAs have the ability to directly or indirectly affect immune responses by their effects on nearby protein-coding genes or their ability to sponge microRNAs (miRNAs) through a variety of methods [20–22]. However, more investigation is necessary to fully understand their potential in leukemia therapy explicitly. This paper primarily discusses (from 2012 to 2024) the function of ncRNAs in immunological checkpoints and their unique processes in the immunotherapy of hematological malignancies.

Characteristics and functions of non-coding RNAs in hematological malignancies

The eukaryotic genome contains around 98% ncRNAs in its transcription, with around 2% translated into proteins [23, 24]. NcRNAs are functional RNA molecules that control biological functions by focusing on certain key biochemical pathways. MiRNAs, which bind to messenger RNAs (mRNAs) of target genes, can lead to translational repression or mRNA degradation. They can act as oncogenes or tumor suppressors depending on their targets. Long non-coding RNAs (LncRNAs) can influence gene expression through interactions with chromatin-modifying complexes, acting as scaffolds for transcription factors or interfering with their binding. They can also influence mRNA stability, splicing, or translation. Some RNAs, like lncRNAs and circular RNAs, can act as miRNA sponges, sequestering miRNAs and increasing the expression of oncogenes or tumor suppressors (Fig. 1) [17, 25, 26]. They are pivotal in gene expression, RNA maturation, and protein synthesis [27]. Recent evidence indicates that non-coding RNAs, including protein-coding mutations and variations within the non-coding genome, contribute to various cancer etiologies [28, 29]. Compelling evidence shows that transcriptional, post-transcriptional, and translational controls, mediated by multiple non-coding RNAs, exert pleiotropic effects on several aspects of leukemia biology [12]. This has led to identifying and characterizing ncRNAs as biomarkers in hematological malignancies, sparking numerous studies in this field over the past decade [13, 30, 31].

Fig. 1.

The process by which non-coding RNAs influence the regulation of oncogenes and the network of tumor suppressors [17]

MicroRNAs

Non-coding RNAs with a distinct hairpin secondary structure, measuring 19–20 nucleotides in length, are known as miRNAs (Fig. 2) [12, 32]. Since its discovery in Caenorhabditis elegans, the species has been detected in the majority of eukaryotic creatures, including humans [33]. At least 30% of the genes that code for proteins are regulated by miRNAs, which comprise 1–5% of the human genome [34]. There are 940 unique miRNA molecules discovered. Encoded at distinct chromosomal loci, miRNAs are translated by RNA polymerase II into primary miRNA transcripts (pri-miRNAs) and then transformed by DROSHA and DiGeorge Critical Region 8 (DGCR8) into pre-miRNA transcripts [32, 35, 36]. Pre-miRNAs produced in the nucleus are exported into the cytoplasm, and DICER and TRBP (transactivation response element RNA-binding protein) may handle them. MiRNAs have the ability to upregulate target translation and suppress gene expression by attaching to untranslated regions of target mRNAs [37, 38]. Blood, plasma, and urine all contain miRNAs, which function as signaling molecules that move cargo between different cells or organs [39]. MiRNAs are crucial in gene expression regulation, acting as fine-tuners rather than switches [40]. They can target hundreds of different mRNAs and multiple mRNAs, creating complex regulatory networks. This allows miRNAs to modulate entire biological pathways and cellular processes [41]. They can also be involved in regulatory circuits like negative feedback loops, feed-forward loops, and double-negative feedback loops, where two miRNAs reciprocally repress each other's expression [42]. MiRNAs are essential for various cellular processes such as development, differentiation, proliferation, apoptosis, and stress responses [43]. They can be regulated through their own turnover, such as enzymatic degradation, target-directed miRNA degradation, or modifications like uridylation or adenylation [44, 45]. They can also be packaged into extracellular vesicles or bound to proteins, influencing gene expression in distant tissues [46, 47]. Their dysregulation has been linked to diseases like cancer, cardiovascular disorders, and neurodegenerative conditions, making them crucial targets for therapeutic interventions and biomarker development [32].

Fig. 2.

Categorization of noncoding RNAs (ncRNAs) [12]

MicroRNAs can act as oncogenes or tumor suppressors in leukemia [48]. They can be dysregulated, affecting gene expression profiles and cellular processes [12]. Oncogenic miRNAs promote cell proliferation and survival by targeting tumor suppressor genes, while tumor-suppressive miRNAs downregulate, increasing oncogene expression and anti-apoptotic proteins [49, 50]. MiRNAs also influence leukemia stem cell behavior, modulating signaling pathways and impacting the bone marrow microenvironment [51, 52]. Their ability to regulate multiple target genes simultaneously allows them to have significant effects on leukemia cell biology and disease progression [53]. However, there isn’t much research on miRNA molecules' involvement in leukemia, and our knowledge of their precise targets and biological activities is still incomplete [53–55].

Chronic myeloid leukemia (CML)

Resistance to Imatinib has been identified as a significant challenge in treating CML [56]. MiRNAs play a major role in the development of drug resistance and tumors, including CML [57]. According to recent research, the miR-221-STAT5 pathway is critical in regulating how sensitive CML cells are to the medication imatinib [58, 59]. MiR-214 alters ABCB1 expression, which has been connected to imatinib resistance in CML patients [60]. MiR-30e inhibits the translation of ABL protein by targeting ABL mRNA. In K562 cells, elevated expression causes apoptosis, suppresses growth, and heightens susceptibility to imatinib therapy. Although miR-203 expression is downregulated in bone marrow, it increases the susceptibility of CML patients to imatinib [61, 62].

Chronic lymphocytic leukemia (CLL)

Venetoclax is a prime example of ncRNAs' crucial role in creating new anti-cancer drugs [63]. This medication makes up for the lack of miR-15a/miR-16-1 targeting B-cell lymphoma-2 (BCL-2) in CLL. It is strong, selective, and well-tolerated [64]. In order to address the absence of miR-15a/miR-16-1 targeting of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in CLL, cercozumab was created [65]. As seen by the synergistic impact on patient-treated CLL cells, the medicines can be used to target distinct pathways in CLL dysregulated by the same driving event, miR-15a/miR-16-1 loss [53]. According to the study, leukemic cells characterized by overexpression of BCL-2 and ROR1 genes, coupled with reduced expression of microRNAs miR-15a and miR-16-1, demonstrate enhanced therapeutic efficacy when subjected to combination treatment utilizing venetoclax, a BCL-2 inhibitor, and cirtuzumab, an anti-ROR1 monoclonal antibody [66]. Drug development relies heavily on microRNAs, as seen by the discovery of the monoclonal antibody ianalumab, which inhibits the production of miR-155 to prevent cancer [67]. In CLL, miR-155 overexpression is carcinogenic, and signals from the microenvironment, such as B-cell activating factor (BAFF) binding to its receptor on the cell surface of the disease, can cause dysregulation of miR-155 [68, 69]. The miR-17/92 polycistronic miRNA cluster, which includes miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a, is overexpressed in lymphoid malignancies such as CLL [70, 71]. A study using transgenic mice overexpressed miR-17/92 in B-cells showed it can function as an oncogene in leukemia development. 80% of these mice developed a B-cell malignancy with increased CD19⁺ B cells [72]. The mechanism triggering miR-17/92 expression is not fully understood. Still, Studies show that MYC induction occurs prior to miRNA up-regulation in unmutated immunoglobulin heavy-chain variable region gene (IGHV) CLL, suggesting a relationship between MYC, breakpoint cluster region protein (BCR) activation, and miR-transcription in CLL [31].

Acute myeloid leukemia (AML)

MicroRNAs have the ability to function as tumor suppressors or oncogenes. BCL-2-associated transcription factor 1 is the target of miR-194-5p, and imbalances between the two are commonly seen in AML patients [73]. Relapsed instances of AML have been reported to have overexpressed MiR-10a-5p [74, 75]. MiR-96 expression is associated with the leukemic load and declines in newly diagnosed AML patients [76, 77]. Tumor-suppressive miRNAs are downregulated in the majority of cancers, whereas oncogenic miRNAs are increased [78]. Hematopoietic stem cells from AML patients with FLT3 internal tandem duplication (FLT3-ITD) and nucleophosmin (NPM1) gene mutations had elevated levels of MiR-155 [79, 80]. It has been proposed that aberrant expression of miR-155 targets two important factors in granulopoiesis, SHIP1 and CEBPB, in individuals with AML [81, 82]. MiR-133 objectives leukemic cells with ecotropic viral integration site 1 exhibit increased drug sensitivity, indicating possible therapeutic targets for leukemias overexpressing ecotropic viral integration site-1 (EVI1) [83, 84]. According to the study, elevated miR-223 expression in AML cell lines both promotes and inhibits cell death and inhibits cell motility and proliferation [85]. In pediatric AML patients, there has been a decrease in MiR-370 expression, which may act as a non-invasive diagnostic and prognostic indicator for the course of the illness [86]. According to the study, by inhibiting ATM, miR-181a upregulation can improve cell cycling and proliferation in the leukemic cell lines HL60 and NB4 [87]. In HL60 cell lines, transfection of MiR-128 has been shown to increase drug sensitivity and apoptosis [88]. While MiR-128 is elevated and overexpressed in a number of cancers, AML cells with NPM1 mutations exhibit a reduction in its expression [12].

Acute lymphoblastic leukemia (ALL)

In patients with high-risk early T-cell precursor ALL (ETP-ALL), miR-221 and miR-222 are increased, which may block ETS1 expression and contribute to the myeloid nature of ETP-ALL [89]. In a mouse model of neurogenic locus notch homolog protein 1 (Notch1)-induced T-cell ALL (T-ALL), miR-19 is the most highly expressed component, and it enhances lymphocyte survival and starts leukemogenesis [90]. In ALL, miR-223, which targets forkhead box protein O1 (FOXO1), increases cell apoptosis while reducing cell invasion, migration, and proliferation [91]. Significant upregulation of the E3 ligase FBXW7 (F-Box And WD Repeat Domain Containing 7) has been seen in T-ALL, which may have a role in the development of Notch1-driven leukemia [92]. MYC (mostly referred to as c-Myc) transcriptionally suppresses the expression of MiR-30a, a microRNA linked to T-ALL, which prevents NOTCH1 and NOTCH2. T-cells are also impacted by the tumor suppressor miR-146b-5p, which is suppressed by TAL1 [93]. The microRNA miR-142-3p, which plays a crucial role in hematopoietic stem cell function, exhibits significantly elevated expression levels in ALL samples. This overexpression is particularly pronounced in pediatric T-cell ALL cases, where patients typically face poorer prognoses compared to healthy donor T-cells [94, 95]. By encouraging leukemic cell proliferation, miR-142-3p causes glucocorticoid treatment resistance in T-ALL. This miRNA is essential for both proliferation and chemo-resistant characteristics [96]. When overexpressed in mouse bone marrow cells relative to B-ALL patient samples, miR-196b, a miRNA overexpressed in T-ALL patient samples, co-expresses with HOXA cluster genes and improves proliferative potential and survival [97, 98]. A tumor suppressor called miR-26b targets PIK3CD (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Delta), which in turn suppresses the PI3K/AKT pathway [99]. Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) plays a role in controlling Ikaros isoforms, which in turn promotes miR-26b expression [100]. MiR-181a increases pre-T cell receptor (pre-TCR) signals and decreases Notch's negative feedback to augment Notch’s oncogenic signals in T-ALL [101]. Comparing patients with multiple medication resistance to those without resistance, the expression levels of miR-326 are considerably lower in the former group [102]. According to recent studies, leukemia patients with the ETV6-RUNX1⁺ fusion gene had higher cell survival and lower apoptosis when the miR-125b-2 cluster was overexpressed. This finding raises the possibility of treating pediatric ALL [103]. By improving our understanding of the processes behind leukemias, the work helps physicians understand the function of miRNA and inform treatment plans.

Long non-coding RNAs

Transcripts longer than 200 nucleotides that are present in differentiated tissues or particular cancer types are known as lncRNAs (Fig. 2) [12, 29]. RNA polymerase II or RNAP III and single-polypeptide nuclear RNA polymerase IV are the transcription factors for lncRNAs, which control gene expression at several cellular levels. They can be bidirectional, sense- or antisense-oriented, intronic, and intergenic [104]. They conduct RNA editing processes, show transcriptional activation patterns, and have traits like those of mRNA transcription [105]. With some functioning as oncogenes and others as tumor suppressors, lncRNAs are essential for RNA processing, nuclear organization, and the control of gene expression. They also regulate gene expression at different levels and are involved in cellular processes such as cell cycle, tumor invasion, and metastasis [105, 106]. By promoting DNA methylation and histone modification, as well as the recruitment of chromatin remodeling complexes to certain genomic regions, they play a critical role in epigenetic modifications [107, 108]. Numerous human illnesses, including several cancers, have been connected to the dysregulation of lncRNAs [109–111]. Even though the activities of miRNAs have been well studied, research on lncRNAs is still developing, and their involvement in hematological malignancies remains mysterious [112, 113]. Leukemia has been shown to exhibit distinct lncRNA expression patterns, suggesting that lncRNAs may be useful as new biomarkers and therapeutic targets [114–116].

CML

Nuclear-enriched autosomal transcript1 (NEAT1) expression is reduced in CML, and its suppression increases imatinib-induced apoptosis [117]. LncRNA-IUR1, a key counteractive regulator of Bcr-Abl-induced carcinogenesis, was barely produced in Bcr-Abl-positive CML cells [118]. The expression of HOTAIR has been associated with the clinical-pathological prognostic classification in AML, indicating that it may be a useful marker and prospective target for future treatment of AML and CML [119]. AML protein HOTAIR reduces the number of colony-forming cells, triggers apoptosis, and stops cell division, all of which lead to CML's imatinib resistance, particularly in those with high MRP1 expression and K562-imatinib-resistant cells. Reducing HOTAIR increases the body's susceptibility to imatinib treatment [120]. LncRNA CCAT2 has been found to be a putative marker for CML diagnosis that may also be used to predict how well a patient would respond to imatinib [121]. Compared to normal cells and CML patients, K562-imatinib-resistant cells had higher levels of the LncRNA SNHG5. In K562-imatinib-resistant cells, overexpression promotes resistance, whereas knockdown decreases resistance [122]. It was discovered that K562 cells had an increased level of metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and that suppressing it inhibited cell division and the cell cycle by focusing on miR-328 [123]. According to recent studies, the lncRNA MALAT1/miR-328 pathway may enhance CML cell growth and resistance to imatinib, suggesting new options for CML treatment [124]. According to the study, chromatin interaction allows lncRNA human meiotic recombination hot spot locus (Hmrhl) to control the expression of genes associated with malignancy in chronic myelogenous leukemia [125].

AML

ZNF571-AS1, a lncRNA, has been identified as a significant player in AML, potentially influencing Janus kinase (JAK), Signal transducer and activator of transcription 5A (STAT 5A), and KIT according to correlation studies [126]. Another lncRNA, LNC00899, has shown substantial promise as a predictive and diagnostic marker for AML [127]. The tumor suppressor MEG3 (Maternally Expressed 3) is associated with a notably poor survival rate in AML and is linked to human cancers [128, 129]. Three lncRNAs have been found as predictive variables for AML risk: lncRNAs AC008753.6, CES1P1, and RP11-342 M1.7. This research used complete lncRNA expression profiling [130]. AML progression is mostly controlled by the LncRNA LINP1 via the HNF4alpha/AMPK/WNT5A signaling pathway [131]. It has been discovered that the lncRNA CRNDE promotes cell cycle progression, proliferation, and apoptosis suppression in the U937 cell line, especially in AML patients [132]. LncRNA PVT1 has been connected to tumor stage, poor prognosis, and carcinogenesis in a number of malignancies [133] and was found to be upregulated in the bone marrow and peripheral blood mononuclear cells of ALL and AML patients compared to healthy individuals [134, 135]. Elevated apoptosis, a G0/G1 arrest in the cell cycle, reduced proliferation, and lower c-Myc protein stability were the outcomes of PVT1 (Plasmacytoma Variant Translocation 1) suppression in Jurkat cells [136]. It has been discovered that lncRNA TUG1, a gene that is overexpressed in AML cells, modulates cell proliferation by decreasing apoptosis and promoting cell proliferation in vitro [137]. As an endogenous RNA that competes with miR155, CCAT1 promotes cell division and suppresses myeloid cell differentiation [138]. LncRNA NR-104098 suppressed EZH2 (Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit) transcription by interacting with E2F1, substantially reducing AML proliferation and promoting differentiation [139]. LncRNA CCD26 was elevated in ALL and AML patients [140]. Higher expression of LINC00265 in the blood and bone marrow of AML patients suggests that this gene may serve as an AML prognostic biomarker. Suppression increased apoptosis and decreased cell invasion, migration, and proliferation [141]. The lncRNA NEAT1, which plays a crucial role in the formation of paraspeckles within the nucleus, was observed to be significantly downregulated in de novo acute promyelocytic leukemia (APL) samples when compared to samples from healthy donors. Further investigation provided evidence that this repression of NEAT1 expression was mediated by the PML-RARα fusion protein. Moreover, during the process of all-trans retinoic acid (ATRA)-induced differentiation of NB4 cells, a substantial upregulation of NEAT1 was noted. Importantly, when NEAT1 expression was reduced through the application of small interfering RNA (siRNA), the ATRA-induced differentiation process was inhibited. These findings collectively suggest a potential regulatory role for NEAT1 in APL pathogenesis and treatment response [142].

ALL

In pre-B ALL patients, overexpression of the genes BALR-1, BRL-6, and LINC0098 is associated with cytogenetic abnormalities and survival rates in B-ALL [143]. In comparison to pre-B-cells (CD10⁺CD19⁺) derived from human cord blood, it was observed that BALR-1 and LINC00958 exhibited heightened expression in ETV6-RUNX1 subtypes. Notably, an increase in BALR-2 expression was detected in patients with MLL rearrangements and those carrying either the t(4;11) or t(9;11) translocations. To explore the functional implications of newly discovered B-ALL lncRNAs, Ouimet and colleagues employed siRNA to reduce the levels of 5 candidate lncRNAs identified in their study [144, 145]. The study discovered that in human NALM-6 leukemia cells, lowering lncRNA RP11-137H2.4 reduced cell migration and proliferation and restored glucocorticoid sensitivity in previously resistant cells. Using the non-leukemic cell line GM12878 as a comparison, Gioia and colleagues examined three downregulated lncRNAs (RP-11-624C23.1, RP11-203E8, and RP11-446E9) in B-ALL further [146]. The lncRNA RP11-137H2.4 has a major effect on cell migration, proliferation, and apoptosis. By inhibiting it, resistant B-ALL cells can undergo apoptosis by regaining an NR3C1-independent glucocorticoid response [145]. Both T-ALL and B-ALL patients showed a decrease in linc-PINT expression [147]. Differential expression of wild-type and mutant NOTCH1 was seen in T-ALL cells for LUNAR1 and lnc-FAM120AOS-1 [148]. Through the HOXA3/EGFR/Ras/Raf/MEK/ERK pathway, the LncRNA HOXA cluster antisense RNA2 (HOXA-AS2) has been shown to reduce glucocorticoid sensitivity in acute lymphoblastic leukemia [149, 150]. In children with ALL, dysregulation of miR-335-3p, which is controlled by the lncRNAs NEAT1 and MALAT1, is associated with a poor prognosis [151].

Circular RNAs

Circular RNAs (circRNAs) constitute a significant category of regulatory transcripts, predominantly originating from protein-coding exons [152]. CircRNAs are produced in a tissue- or developmental stage-specific way, form closed-loop structures, and show relative stability in the cytoplasm [153]. CircRNAs are RNA viruses that are usually created at low levels by co-transcription from mRNA. They have several exons in their structure and are controlled by cis and trans-acting elements. Through alternative splicing, a single gene can produce several circRNA isoforms by including or deleting internal introns [154]. CircRNAs play critical physiological and functional roles in regulating gene expression, as evidenced by a recent study. By serving as spies for microRNA binding, decreasing their cellular availability, and upregulating target mRNAs, circRNAs contribute to post-transcriptional control [155, 156]. It has been discovered that circular RNAs are changed in a number of clinical situations, suggesting a possible function for them in human illnesses, including cancer [157]. A miRNA intermediate is frequently involved in the connection between circRNAs and a number of human disorders, including cancer, according to recent studies [158–160]. CircRNAs, which are more prevalent in blood than linear mRNAs, have been shown in research to have potential use as new biomarkers in routine clinical blood samples [12, 161].

CML

Researchers Pan and colleagues discovered a link between resistance to Imatinib, a second-generation tyrosine kinase inhibitor (TKI), and f-circRNA circBA9.3, which is derived from BCR-ABL1 mRNA. In TKI-resistant patients, they discovered higher circBA9.3 levels; in BCR-ABL-negative cell lines, they discovered enhanced cell proliferation and cancer development. Additionally, they discovered a positive association between BCR-ABL1 expression and circBA9.3 levels, with high circBA9.3 expression correlated with lower apoptosis [162]. Hsa_circ_100053 levels were discovered to be higher in CML patient cells and serum by Ping et al.'s investigation, indicating that it may be a possible biomarker for CML. Elevated expression was linked to BCR/ABL1 mutant status, advanced clinical stage, and imatinib resistance. Furthermore, it was suggested that elevated hsa_circ_100053 levels were a poor predictor of overall survival in CML patients [163]. In a separate study, Liu and colleagues [164] identified hsa_circ_0080145 as up-regulated in CML patients cells and in K562 and KU812 cell lines through an RNA-sequencing screen. The research discovered that by acting as a sponge for miR-29b, hsa_circ_0080145 silencing inhibited leukemic cell growth. These genes were implicated in the biosynthesis of heterocycles, the cAMP signaling pathway, and the systemic lupus erythematosus pathway. This is consistent with a prior work that discovered that overexpressing miR-29b in K562 cells and downregulating it in CML prevented leukemic cell proliferation and encouraged apoptosis [165]. Recent investigations have revealed circRNAs as putative drug resistance indicators, prospective therapeutic targets, and potential biomarkers for CML.

CLL

It has been discovered that the circRNA circulating factor beta (circCBFB) is up-regulated in untreated CLL cells, indicating that it may serve as a prognostic and diagnostic marker for CLL patients. The Wnt/ß-catenin pathway is activated by circulating factor beta, and this can result in a proliferative and anti-apoptotic phenotype that aids in the development of CLL. Low overall survival and a shorter survival duration are linked to high levels of circCBFB and circRPL15, respectively [166]. Because circ_0132266 may bind to miR-337-3p in CLL cell lines, Wu et al. suggested it as a possible tumor-suppressor in CLL. MiR-337-3p's primary target, PML, controls both gene expression and cell survival. MiR-337-3p levels may rise in response to circ_0132266 reductions, perhaps displaying tumor-suppressive characteristics in CLL [167]. Tests have been conducted on the circular RNA circRPL15 as a possible diagnostic biomarker in CLL patients, particularly those without an IGHV mutation. Increased RAF1 protein levels brought on by circRPL15 upregulation trigger mitogen‑activated protein kinase (MAPK) signaling and encourage cell proliferation. This theory was supported by the reduction in mitogenic factor phosphorylation seen in human cell lines following circRPL15 knockdown [168].

AML

Over the past few years, a growing number of dysregulated circRNAs have been identified in AML. For instance, Chen et al. [169] discovered that via sponging microRNAs in the miR-181 family, which control hematopoietic differentiation, circANAPC7, an up-regulated circRNA, may be linked to AML. Moreover, they discovered that circ_0009910 is a circRNA that increases the development of cancer by sponging up the tumor-suppressor miR-20a-5p [170]. Fan et al. [171] found that circ_100290 increased AML cell proliferation and inhibited apoptosis by sponging miR-293, thereby increasing Rab10 expression, a member of the oncogenic RAS family. Circ_0004277 levels were discovered to vary across AML patients by Li et al. [172]; low levels were observed in initially diagnosed patients, but they were restored during induction treatment. Nevertheless, during relapse, levels dropped, indicating promise as a predictive and diagnostic biomarker. Circ 0004277 may be a component of a complex network comprising mRNAs and miRNAs, according to a bioinformatic study. In cytogenetically normal AML (CN-AML) cell lines, Hirsch and colleagues found numerous circRNAs originating from NPM1, with circRNA hsa_circ_0075001 being more abundant in AML cells [173]. Forty-six undifferentiated blast patients had larger quantities of circular RNA, which had an inverse relationship with the expression of genes influencing the differentiation of hematopoietic cells [174–176]. Patients with high hsa_circ_0075001 levels showed a drop in the abundance of the miR-181 target gene, which may be related to circRNA sequestration by NPM1, which has miR-181 binding sites [173]. It's significant to remember that miR-181 is essential for both cellular differentiation and the emergence of hematological cancers [177]. In pediatric Acute Myeloid Leukemia patients, Yuan and colleagues detected a rise in circ_0004136, indicating that it may promote cell proliferation by binding to and suppressing miR-142, a microRNA associated with pediatric AML [178]. In another investigation, Yi et al. found that circVIM, which is produced from the VIM (vimentin) gene, was present in 113 AML patients [179]. Elevated circVIM levels in AML are associated with reduced leukemia-free survival and overall survival, indicating that it may serve as a prognostic indicator. Circulated circRNA, circDLEU2, inhibits the action of miR-496 and encourages the growth of tumors in mice. Elevated circDLEU2 levels have been shown to upregulate PRKACB expression, which may alter cell apoptosis and proliferation [180]. Elevated levels of the circular RNA circKLHL8 have been associated with better overall outcomes, longer survival times between events, and a decreased proportion of malignant blasts in bone marrow and blood [181]. It was proposed that two more circRNAs, circFOXO3 and circFBXW7, function as tumor suppressors in AML [181, 182]. APL-derived NB4 cells treated with ATRA showed differential expression of a number of circRNAs, including circHIPK2, circHIPK3, circPVT1, circRELL1, and circSMARCA5. Li and colleagues discovered these circRNAs. CircHIPK2 rose with complete remission and fell in those who had just received a diagnosis. Its association with cell maturation was important because it upregulated the transcription factor CEBPA, which is involved in hematopoiesis, and sponged miR-124-3p [183]. Up-regulated circRNAs in extramedullary infiltration (EMI) bone marrow samples may affect signal transduction, migration, and cell adhesion, according to research by Lv et al. [184]. According to the study, hsa_circ_0004520 could control VEGFA expression, which might encourage angiogenesis in AML-EMI. The design of AML treatment is complicated by the intricacy of cytogenetic and molecular abnormalities. Resistance may eventually arise even with cytarabine and anthracycline induction treatment [185]. Sun et al. discovered that circMYBL2 knockdown reduced cell proliferation in animals in culture while increasing the susceptibility of human FLT3-ITD⁺ cells to TKI quizartinib. CircMYBL2 improved FLT3 translation by enabling PTBP1-mediated mRNA binding [186]. Shang et al. discovered that circPAN3 plays a significant role in AML cell lines' doxorubicin resistance. By binding miR-153-3p and miR-183-5p, CircPAN3 modifies the expression levels of XIAP. Drug sensitivity was restored upon downregulation of circPAN3, indicating circPAN3's involvement in AML resistance to traditional chemotherapies [187].

ALL

Studies on circRNAs revealed that circRNAs accounted for more than 10% of transcripts in hematopoietic stem cells and naïve B cells, suggesting that circRNAs express differently in acute lymphoblastic leukemia [188]. Various circRNAs produced from the partner fusion gene AF4 of Mixed Lineage Leukemia (MLL) were found in a 2019 research by Huang et al. [189]. Elevated circAF4 levels were discovered in leukemia cell lines and in patients less than eight years old. The degree of the illness was shown to be correlated with Circaf4 levels, and in cells that had the MLL-AF4 translocation, silencing Circaf4 enhanced apoptosis. In mice, circAF4 knockdown decreased spleen infiltration and increased survival. Circaf4 may bind to miR-128-3p and sequester microRNA from the fusion MLL-AF4 mRNA, allowing MLL-AF4 to be expressed. The overexpression of miR-128-3p in vivo and the silencing of circAF4 supported this regulatory axis. According to a 2019 study by Dal Molin et al., [190] some rearrangements between MLL and other genes may also lead to the synthesis of disease-associated aberrant circRNAs in addition to producing alternate isoforms of circRNAs in various leukemia subtypes. According to Hu et al.'s 2018 study, [191] dysregulation of circPVT1 in ALL cell lines increased the expression of MYC and BCL2, sponging miR-125 and let-7 and controlling the let-7 family members' ability to operate as tumor suppressors. These effects were seen to enhance cell proliferation and prevent apoptosis [192]. Using bioinformatics, Gaffo et al. detected and measured circRNAs in T cells, B cells, and monocytes. Depending on the cell type and differentiation stage, the signatures changed. Examination of circRNAs that were differently expressed in pediatric ALL patients' B-cell progenitors revealed that the transcription factors BSAP are encoded by circPVT1, circHIPK3, and circPAX5. These circRNAs are up-regulated. CircPAX5 and CircHIPK3 binding to miR-124-5p may work in concert to obstruct B cell development and advance the course of the illness [193].

Immune checkpoints and hematological malignancies

Programmed death-1 (PD-1)

The transmembrane protein PD-1, also known as CD279, was initially identified by Ishida et al. in 1992 during a search for apoptosis-inducing genes [194]. The 288 amino acid protein, which is produced by the PDCD1 gene, has an intracellular domain with an ITSM and ITIM immunoreceptor tyrosine-based motif, a transmembrane domain, and a single V-like domain [195, 196]. Not found in resting T cells, PD-1 is expressed in a variety of immune cells such as effector T cells, regulatory T cells, naïve and activated B cells, natural killer cells, myeloid dendritic cells, and monocytes [197]. These immunological checkpoints are frequently used to cover a variety of tumors, including hematologic cancers [198, 199]. Tumor-infiltrating lymphocytes (TILs) can provide extrinsic or intrinsic signals that activate PD-L1 or PD-L2 in tumor cells, which in turn induce immunological escape signals [200, 201]. Large B cell lymphomas that are rich in T cells and histiocytes (TCHRBCLs) are distinguished by a dense population of CD8⁺ T cells and histiocytes, together with a small number of malignant B cells [202]. The expression of PD-L1 in TCHRBCL is diverse at the interface between malignant B cells and the inflammatory background. It is particularly robust in the histiocytes that are in close proximity to lymphoma cells, indicating that immune escape signals are influenced by both tumor and background inflammatory cells [203]. Four mechanisms have been reported for the expression of PD-L1 in lymphoid neoplasms: copy number alterations, translocations involving 9p24.1/PD-L1/PD-L2, and overexpression in tumor cells of classical Hodgkin lymphoma, primary mediastinal large B cell lymphoma, Epstein-Barr virus-negative PCNSL, primary testicular lymphoma, and a subset of diffuse large B cell lymphoma. These mechanisms account for the majority of PD-L1 expression in lymphoid neoplasms [204–208]. According to a study, all cases of EBV-positive diffuse large B-cell lymphoma (DLBCL) and DLBCL linked to EBV immunodeficiency express PD-L1 [203]. Other EBV-related lymphoproliferative illnesses, such as plasmablastic lymphoma, primary effusion lymphoma, extranodal NK/T cell lymphoma, and EBV⁺ post-transplant lymphoproliferative disorder, all express PD-L1 [203, 209, 210]. Preclinical research indicates that the PD-1 pathway is dysregulated in AML. The murine leukemic cell C1498 exhibits increased PD-L1 expression in vivo, indicating that the leukemic cells' milieu supports PD-L1 expression [211]. After being inoculated with C1498, regulatory T cells (Tregs) and CD8⁺ T cells expressing PD-1 increased in the liver, where C1498 leukemic cells spread [212]. This finding is also observed in the bone marrow of AML patients [213]. Tregs limit IFN-γ release and CD8⁺ T cell proliferation; nevertheless, in animals lacking PD-1 or in mice given anti-PD-L1 antibody injections, their suppressive function is compromised [212]. When compared to wild-type mice, PD-1 KO animals that were implanted with C1498 leukemia cells had a greater anti-tumor response and a longer life time [211, 212]. The in vivo administration of anti-PD-L1 antibody to C1498-challenged wild-type mice demonstrated similar anti-tumor activity [211, 212]. Clinical evidence shows that AML patients have a dysregulated PD-1 pathway, with much greater T cell PD-1 expression than in healthy persons [214].

Cytotoxic T-lymphocyte antigen-4 (CTLA-4)

CTLA-4, also known as CD152, is a protein that was initially discovered by Brunet et al. [215]. This protein is encoded by the CTLA-4 gene, which is located on chromosome 2q33.2 and consists of four exons. The protein has a single V-like domain with ligand binding sites and is a member of the immunoglobulin superfamily [216, 217]. The CTLA-4 protein is composed of 223 amino acids and has a calculated molecular weight of 24.6 kDa. After activation, one to two days later, naïve resting T cells will exhibit surface expression of CTLA-4, which is mostly located in the cytoplasm [218, 219]. Conversely, memory T cells exhibit a rapid induction of CTLA-4 expression upon activation, and this expression persists longer than in naïve resting T cells [220]. Notably, CTLA-4 is constitutively expressed in regulatory T cells [221]. In patients with peripheral T-cell lymphoma, mycosis fungoides, and Sézary syndrome, there is an observed upregulation of CTLA-4 expression. However, this is not the case in B-cell lymphoma [222–224]. A rearrangement between CTLA4 and CD28 is evident in a subset of patients with various conditions, including angioimmunoblastic T-cell lymphoma, extranodal NK/T-cell lymphoma, peripheral T-cell lymphoma (not otherwise specified), Sézary syndrome, and adult T-cell leukemia/lymphoma [225–228]. A fusion protein produced by the rearrangement of CTLA4 and CD28 enhances T-cell signaling through the MAPK and AKT pathways [226]. Research has indicated that CTLA-4 contributes to the immune evasion of AML and that CTLA-4 inhibition improves the ability of cytotoxic T-lymphocytes to destroy any remaining leukemic cells [229]. The CTLA-4 polymorphism CT60 AA genotype, found in the 3′-UTR of the CTLA-4 gene, has been linked to relapse in AML patients [230].

Lymphocyte activation gene-3 (LAG-3)

In the wake of the clinical success achieved by targeting co-inhibitory molecules CTLA-4 and PD-1, other molecules of the same class, namely LAG-3, have garnered increased attention. The lymphocyte activation gene-3 (LAG-3, also known as CD223) was first identified by Triebel and colleagues in 1990 [231]. This gene, located at 12p13.31, is composed of eight exons and encodes a protein of 498 amino acids. Structurally, LAG-3 bears a resemblance to CD4, comprising one immunoglobulin-like V-type domain and three immunoglobulin-like C2-type domains. The intracellular domain of LAG-3 contains a unique KIEELE motif, which plays a crucial role in modulating T-cell activity [232]. LAG-3 expression is observed in activated T cells, NK cells, activated B cells, and plasmacytoid dendritic cells [231, 233, 234]. LAG-3 acts as a negative regulator of CD4 and CD8 T cell expansion both in vitro and in vivo [235]. The exact mechanisms behind this regulation remain unclear, but the co-expression of LAG-3 and PD-1 in TILs in mouse models and human tissue suggests a similar role for LAG-3 [236–238]. Research has indicated that when both PD-1 and LAG-3 are simultaneously inhibited, CD8⁺ T cells' anti-tumor activity is increased as opposed to when only one molecule is targeted [236, 237]. In the context of Hodgkin lymphoma (HL), it has been observed that an elevated presence of LAG-3 on TILs and peripheral blood lymphocytes is linked to the suppression of Epstein-Barr Virus (EBV)-specific T-cell-mediated immunity. Notably, compared to HL patients in remission, the number of CD4⁺ LAG-3 circulating T-cells was considerably greater in patients with active illness [239]. A particular study highlighted that in the Tumor Microenvironment (TME) of classical HL, LAG-3 is almost invariably co-expressed [240]. LAG-3, which is mostly expressed on PD-1⁺ T-cells, is overexpressed in follicular lymphoma (FL) and is associated with worse outcomes. IL-12 causes this upregulation, which eventually wears down T cells. The enhancement of CD8⁺ T-cell activity via blocking both PD-1 and LAG-3 leads to a rise in IL-2 and IFN-γ production [241].

T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3)

Hepatitis A virus cellular receptor 2 (HAVCR2), also known as TIM-3, was initially discovered by Monney et al. in 2002 [242]. The HAVCR2 gene, located at 5q33.3, encodes TIM-3 and comprises seven exons. The transmembrane protein TIM-3 has a cytoplasmic tail, immunoglobulin-like V-type domain, mucin domain, and signal peptide sequence [242]. It is expressed in various immune cells, including cytotoxic T cells, T helper 1 cells, regulatory T cells, NK cells, monocytes, and dendritic cells. TIM-3 has multiple ligands, such as galectin-9, high mobility group protein B1 (HMGB1), and phosphatidyl serine [243, 244]. It is still unclear how exactly the tyrosine residues in TIM-3’s cytoplasmic domain interact with downstream signaling pathways. Studies have shown that targeting TIM-3 can significantly enhance anti-tumor activity in tumor mouse models [245]. Moreover, it has been demonstrated that concurrent suppression of TIM-3 and PD-1 increases the anti-tumor efficacy of CD8⁺ TILs [246].

B7-H3/CD276

The B7 family, recognized as a collection of immune regulatory ligands, plays a crucial role in the modulation of T lymphocyte activation and differentiation. They are widely distributed in both innate and adaptive immune cells, as well as different cancer tissues. They are closely related to the CD28 superfamily (Fig. 3) [247]. This enhances cancer's capacity to elude the immune system [248]. The B7 family is composed of ten members, including the well-studied B7-H1 (PD-L1), B7-1 (CD80), B7-2 (CD86), B7-DC (PD-L2), B7-H2 (CD275), B7-H3 (CD276), B7-H4, B7-H5, B7-H6, and B7-H7 (HHLA2) [249]. B7-H3, a member of the B7 family, has been the focus of considerable attention since its discovery in 2001 [250]. Through a variety of pathways, B7-H3 has a paradoxical function in T cell activation, contributing to carcinogenesis, metastasis, and malignant behaviors. A worse prognosis is associated with its expression. A thorough analysis of B7-H3 is required to further our knowledge of cancer immunotherapy and spur clinical development. The function, underlying mechanisms, expression, involvement in various cancer types, and advancements in B7-H3 immunotherapy in clinical trials are all outlined in this review of current research [247]. The type 1 transmembrane glycoprotein human B7-H3 gene, located at 15q24.1, encodes 316 amino acids through 12 exons and has two isoforms: 2IgB7-H3 (B7-H3 VC) and 4IgB7-H3 [250, 251]. In humans, 4IgB7-H3 is the primary isoform expressed on immunocytes and malignant cells [252]. The 93% amino acid similarity between the structure of the human 2IgB7-H3 and the mouse B7-H3 gene, which is found on chromosome 9, is noteworthy [253]. The FG loop of the IgV domain is essential to the inhibitory activity of the murine B7-H3 protein, according to its crystal structure [254]. In addition to its transmembrane form, soluble B7-H3 (sB7-H3) has been detected in normal human blood [255]. sB7-H3 is produced by alternative splicing from the fourth intron of B7-H3 [256] or matrix metallopeptidase (MMP) [255], and serum levels of sB7-H3 have been linked to prognosis in a number of cancers [257, 258]. Primary AML blasts and monocytic AML cell lines both exhibit significant levels of B7-H3 expression. Its CAR-T has shown strong anti-AML properties both in vitro and in a xenograft form [259].

Fig. 3.

Presently recognized immune checkpoint receptors along with their corresponding ligands [247]

Immune checkpoints and ncRNAs

NcRNAs are essential for controlling immunological checkpoints and other cellular processes. Trans-membrane CTLA-4, which is expressed on CD4⁺ and CD8⁺ T cells, binds to CD80 and CD86 to block T-cell signaling. It has been determined that there are two miRNAs that target CTLA-4, with MiR-138 acting as a tumor suppressor in cancer. Through its binding to PD-1 and CTLA-4, MiR-138 inhibits the development of glioma cells in vivo [260]. Conversely, by preventing CTLA-4 expression on T cells, MiR-155 stimulates anti-cancer immune responses; its overexpression may improve immunotherapy [261].

miR-155

MicroRNA-155 (miR-155) is linked to B-cell lymphoma progression by enhancing the interaction between B-lymphoma cells and CD8⁺ T cells in the tumor microenvironment, potentially inhibiting the PD-1/PD-L1 pathway [262]. MiR-155 transgenic mice displayed pre-B-cell proliferation in the spleen and bone marrow and exhibited malignant B-cell transformation [263]. MiR-155 enhances PD-L1 expression in B-lymphoma cells by binding to the 3′-UTR, promoting CD8⁺ T-cell apoptosis and maintaining tumor immunity in a PD-1/PD-L1-dependent manner [262]. The AKT and ERK pathways are crucial regulators of PD-1-mediated CD8⁺ T-cell function [264]. Overexpression of miR-155 in DB cells increased their sensitivity to anti-PD-L1 and anti-PD-1 antibodies [262]. In nude mice, downregulation of miR-155 induced apoptosis in B-lymphoma cells and delayed the formation of xenograft tumors [265]. Two inhibitory receptors, miR-155 and BTLA (B- and T-lymphocyte attenuator), may be suitable targets for anti-PD-L1 protein therapy in malignant B-cell neoplasms. When BTLA is coupled to SHP-1 and SHP-2 phosphatase, it becomes phosphorylated and decreases the production of IL-2. Activated Th1 cells produce BTLA [266]. According to Liu J. et al., the elevated protein miR-155 selectively targets the 3′UTR of BTLA, resulting in a 60% reduction in the protein's surface expression in T-activated cells [267].

SNHG14/ZEB1

Zinc finger E-box-binding homeobox gene 1 (ZEB1), a transcription factor, is associated with gene regulation in various cancer cells, influencing invasion, migration, epithelial-mesenchymal transition (EMT), and proliferation [268–270]. The SNHG14 (Small Nucleolar RNA Host Gene 14) regulates invasion, migration, and proliferation, which gives many illnesses chemo-resistance. This function is critical in the development of cancer [271]. Prior research has demonstrated that ZEB1 can improve immune evasion by upregulating PD-L1 expression in cancer cells [272, 273]. When SNHG14 was silenced, ZEB1's RNA expression decreased, which in turn affected ZEB1 and PD-L1's protein expressions in DLBCL cells [271]. SNHG14/ZEB1 promoted DLBCL cells' interaction with CD8⁺ T lymphocytes and triggered death via the PD-1/PD-L1 pathway [271]. This shows that PD-1/PD-L1 and SNHG14/ZEB1 may combine to provide a viable target for DLBCL therapy.

miR-340-5p

KMT5A, a miR-340-5p target, regulates CD8⁺ T cell activity and enhances DLBCL cells' immune system inhibition, thereby influencing tumor development control [274]. Tumor cells expressing CD73 negatively regulate the anti-tumor T-cell response and can also increase T-cell apoptosis [275]. KMT5A silencing boosted CD73's ubiquitination in LY-1 cells, which was subsequently decreased by COP1 knockdown, resulting in CD73's downregulation [276]. In DLBCL cells, the miR-340-5p/KMT5A axis functions as an antitumor mediator independently of immunological modulation [276]. This presents a fresh viewpoint on the management of DLBCL.

miR-21

MicroRNA-21 (miR-21) is a key regulator in the disease progression of B-cell lymphoma [277, 278]. An experiment revealed a pre-B malignant lymphoid-like phenotype caused by miR-21 overexpression [279]. Through the regulation of p-STAT3, MiR-21 upregulates ICOS on Tregs, boosting their interaction with epithelial cells and encouraging tumor formation and chemo-resistance in B-cell lymphoma [280]. These results could offer new approaches to treating B-cell lymphoma.

miR-28

According to recent studies, miR-28 modulates T-cell fatigue by upregulating the expression of checkpoint inhibitor receptors such as PD-1, TIM-3, and LAG3, as well as lowering T-cell secretions of TNF-a and IL-2 [281]. In addition, miR-28 serves as a protective agent against Burkitt lymphoma (BL), and its suppression by MYC plays a role in the development of B-cell lymphoma [12]. By focusing on immunological checkpoints, RNA therapies are thought to be a viable approach to cancer immunotherapy. Nevertheless, more research into the molecular processes and advances in medication development is required to completely comprehend the connection between ncRNA and immunological checkpoints.

Role of checkpoint inhibition in hematological malignancies

ICIs, a type of monoclonal antibody, target immune checkpoints that are frequently overexpressed on cancer cells and cells in the surrounding immune and stromal microenvironment [282]. ICIs have been licensed for use in conjunction with chemotherapy, targeted cancer treatment, and other immunotherapeutic drugs; they have dramatically improved patient prognosis [283–285]. Some cancer types have shown long-term remission with ICI treatment; however, not all patients benefit from this because of tumor immunogenicity and immune microenvironment phenotypic reductions [286–288]. Because of ncRNA's diverse roles and ability to simultaneously block several checkpoint receptors, RNA-based therapies hold great promise for advancements in cancer immunotherapy [289]. Developing treatment approaches, identifying predictive biomarkers of ICI response, and comprehending basic and acquired resistance mechanisms are important obstacles in the field of cancer immunotherapy. Numerous ncRNAs linked to the ICI response have been discovered recently [290, 291]. Classical Hodgkin Lymphoma (CHL) is the most extensively researched lymphoid neoplasm in the context of PD-1 blockade. Nivolumab, a fully humanized IgG4 anti-PD-1 monoclonal antibody, has been shown to have a satisfactory safety profile and significant clinical activity in patients with relapsed or refractory CHL, as evidenced by a phase 1b study (NCT01592370) [292]. Pembrolizumab, a different, completely humanized IgG4 anti-PD-1 monoclonal antibody, has equivalent clinical efficacy to nivolumab and a tolerable safety profile in patients with relapsed or refractory CHL (NCT01953692, KEYNOTE-013) [293, 294]. The clinical activity of pembrolizumab was further substantiated with a multicohort phase 2 study (KEYNOTE-087, NCT02453594), which observed an overall response rate (ORR) of 65–72% with a CR rate of 22% across all cohorts [295, 296]. Primary mediastinal large B cell lymphoma (PMBL), primary central nervous system lymphoma (PCNSL), and primary testicular lymphoma (PTL) are thought to be strong candidates for PD-1 blocking based on the underlying genetic abnormalities. Nineteen patients with relapsed or refractory PMBL were enrolled in an independent cohort in phase 1b research using pembrolizumab (NCT01953692, KEYNOTE-013). The ORR was 41% with a median follow-up of 11.3 months, and 2 and 5 patients, respectively, achieved CR and PR. A global multi-center phase 2 experiment (KEYNOTE-170, NCT02576990) is presently being conducted in response to these findings [297]. Patients with varying ORR (30–40%), such as those with mycosis fungoides/Sézary syndrome (MF/SS), follicular lymphoma, T cell lymphoma, or DLBCL have also tried PD-1 blocking (NCT01592370 and NCT02243579) [298, 299]. Numerous anti-PD-1 antibodies, including as nivolumab, pembrolizumab, AMP-224, BGB-A317, MEDI0680, PDR001, PF-06801591, and REGN2810, are being studied in the context of immunotherapy. The US Food and Drug Administration (FDA) has approved atezolizumab (Tecentriq^®, Genentech), a completely humanized IgG1 anti-PD-L1 monoclonal antibody, to treat metastatic non-small cell lung cancer. According to preliminary findings, individuals with relapsed/refractory DLBCL or FL who receive atezolizumab in addition to obinutumab (an anti-CD20 antibody) show good tolerance and clinical effectiveness (NCT02220842) [300]. Two completely human monoclonal anti-CTLA-4 antibodies are available: tremelimumab (Pfizer) and imipomumab (Yervoy®, Bristol-Myers Squibb). In patients with relapsed or resistant B cell lymphoma, an early pilot study using ipilimumab monotherapy showed a poor overall response rate (ORR) of 11% [301]. The three forms of LAG-3 therapeutics include LAG-3 fusion proteins, LAG-3-targeting monoclonal antibodies, and bispecific LAG-3 antibodies. The majority of LAG-3 therapies are completely humanized IgG4 monoclonal antibodies [302]. It has been shown that LAG-3-targeting monoclonal antibodies decrease the synthesis of both IL-12 and IFN-γ. Additionally, these antibodies block the positive signal that MHC-II sends to monocytes and the T-cell response to IL-12 [303]. Anti-LAG-3 monotherapy might not be the optimal course of action; combination therapies, especially those including PD-1 inhibitors, are being researched. The quantity and functionality of antigen-specific CD8⁺ T cells are increased by blocking PD-1 and LAG-3 [304]. Other monoclonal antibodies targeting LAG-3, such as HLX26 (NCT05078593 and NCT05400265), IBI110 in diffuse large B cell lymphoma (NCT05039658), INCAGN02385 (NCT03538028, NCT04370704, NCT05287113, NCT04586244), Sym022 (NCT03489369, NCT03311412, NCT04641871), and TSR-033 (NCT03250832, NCT02817633), are also under clinical investigation [305]. Ipilimumab, an immunotherapeutic agent, has demonstrated efficacy in patients with AML who have relapsed following allogeneic stem cell transplantation. A phase I/Ib study (NCT01822509) administered ipilimumab at a dosage of 10 mg/kg to patients with relapsed hematologic malignancies post-allogeneic stem cell transplant. This cohort comprised 16 patients with AML, 2 with Myelodysplastic Syndromes (MDS), and 1 with myeloproliferative neoplasm. Out of the 22 patients who received the 10 mg/kg dosage, 5 patients (23%) exhibited a complete response. This included 3 patients with leukemia cutis, 1 with myeloid sarcoma, and 1 with AML. Furthermore, 4 additional AML patients, although not achieving an objective response, displayed a reduction in tumor burden [306]. In a separate phase I study, ipilimumab monotherapy was administered to high-risk MDS patients (n = 11) who had not responded to hypomethylating agents. While no objective responses were reported, disease stabilization was observed in five patients (45%) [307]. Numerous clinical trials are currently investigating the use of anti-CTLA-4 antibodies, either as monotherapy or in combination with other treatments, in patients with MDS or AML (NCT01757639, NCT02117219, NCT02846376, and NCT02890329). Preliminary results from a phase 2 study (NCT02530463) investigating various combinations of nivolumab, ipilimumab, and azacitidine in MDS patients have also been reported [308].

Non-coding RNA-based immunotherapy

The potential of ncRNAs as therapeutic targets for cancer treatment is being explored due to their link with resistance to immunotherapy. A range of immunoregulatory miRNA mimics/antagonists, including miR-26, miR-33a, miR-34, miR-101, miR-125, miR-21, miR-31, miR-32, miR-100, miR-192, and miR-211, are currently undergoing pre-clinical and clinical trials for cancer treatment. However, none have achieved a significant therapeutic breakthrough [309–312]. The application of miRNA mimics/antagonists in cancer treatment is hindered by their physiochemical properties, such as their vulnerability to nuclease-mediated degradation, potential for off-target side effects, and low cellular uptake [313]. Various strategies are being investigated to overcome these challenges. For instance, chemical modifications like locked nucleic acid (LNA), phosphorothioate-containing oligonucleotides, and peptide nucleic acids (PNAs) have been shown to enhance the stability, cellular targeting, uptake, and delivery efficacy of miRNA-based drugs [314]. Furthermore, developing targeted delivery formulations, such as liposomal and polymeric-based delivery platforms, offers a significant opportunity to apply ncRNA-based therapeutics [315].

Drug resistance has been a significant hurdle in the effectiveness of treatment, leading to disease relapse/progression and impacting prognosis. Clinical studies have shown that approximately 30–50% of cancers develop primary or secondary resistance after an initial response to T-cell-based immunotherapy [316–318]. This resistance is potentially due to immune evasion from immune-surveillance, facilitated by alterations in tumor cells and the TME at various levels [319]. Immunomodulatory treatments have shown efficacy in promoting a balanced anti-tumor immune response, activating cytotoxic T lymphocytes (CTLs), and inhibiting tumor growth [320]. However, these approaches are not without limitations, including adverse events and resistance observed in certain types of cancer [321, 322]. Recent focus has shifted towards the role of immunoregulatory ncRNAs in eliciting and monitoring specific immune responses in the context of cancer immunotherapy [323, 324]. Previous studies have highlighted the crucial role of miR-491 in regulating the proliferation and apoptosis of CD8⁺ and CD4⁺ T-cells. This regulation is achieved by reducing the expression of IFN-γ by targeting cyclin-dependent kinase 4, the transcription factor T-cell factor 1, and the anti-apoptotic protein B-cell lymphoma 2-like 1[325]. Circ_0009910 has the potential to inhibit proliferation, sphere formation, and autophagy while promoting apoptosis in AML cells. This is achieved by regulating B4GALT5 expression and activating the PI3K/AKT signaling pathway through the absorption of miR-491-5p. These findings suggest that circ_0009910 could serve as a potential biomarker for AML treatment [326]. Consequently, miR-491 could serve as a potential immunomodulatory biomarker in cancer immunotherapy. Furthermore, miR-381-3p has been shown to induce T-cell differentiation by targeting FOXO1, activating the transcription factors T-bet and RORγt [327]. Also, the reestablishment of ROCK1 (Rho Associated Coiled-Coil Containing Protein Kinase 1) expression negates the suppressive effect of miR-381-3p on cell proliferation, invasion, and migration. This suggests that miR-381-3p functions as a tumor suppressor in pediatric AML by targeting ROCK1 [328]. MiR-381-3p, a key immunomodulatory ncRNA, has the potential to be a therapeutic target for pediatric AML treatment, potentially serving as a predictive biomarker for patient response to such treatments.

Certain lncRNAs, referred to as immune-related lncRNAs, play a crucial role in modulating the T-cell-mediated immune response and the release of inflammatory cytokines. This modulation results in an immunosuppressive TME, which leverages immune checkpoint pathways (Fig. 4) [329]. Studies reveal that different lncRNAs attract and activate cells that suppress the immune system, such as Tregs and myeloid-derived suppressor cells (MDSCs), and these cells are associated with a worse prognosis and resistance to immunological treatment in the TME [330]. In the context of tumor biology, the lncRNA known as lnc-chop plays a crucial role in the regulation of myeloid-derived suppressor cells (MDSCs). Specifically, the suppression of lnc-chop within MDSCs enhances the release of Interferon-gamma (IFN-γ) by both CD4⁺ and CD8⁺ T cells, thereby fostering an immunosuppressive environment. This process is further modulated by the activation of the transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) and the subsequent upregulation of Arginase-1 (Arg-1), Nitric Oxide Synthase 2 (NOS2), NADPH Oxidase 2 (NOX2), and Cyclooxygenase-2 (COX2). This upregulation occurs via the binding of both the C/EBPβ homologous protein (CHOP) and the liver-enriched inhibitory protein (LIP), which are integral to this process. Moreover, lnc-chop augments the production of Nitric Oxide (NO), Hydrogen Peroxide (H₂O₂), and Reactive Oxygen Species (ROS), as well as the expression of Arg-1. This is achieved by promoting the enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in the promoter regions of Arg-1, NOS2, NOX2, and COX2 [331].

Fig. 4.

The control of immune checkpoint molecules in cancer by long non-coding RNAs [373]

According to recent research, modifying lncRNAs may function as an immune sensitizer to counteract the resistance of immunotherapy. For example, lncRNAs that suppress NEAT1 have been shown to decrease CD8⁺ T-cell apoptosis and boost cytolytic activity through the miR-155/Tim-3 pathway [332]. Among the lncRNAs that affect antigen presentation, long intergenic non-coding RNA for kinase activation (LINK-A) is found to be highly expressed in a subset (25%) of triple-negative breast cancer patients. LINK-A appears to negatively regulate the recruitment of Antigen-Presenting Cells (APCs) and CD8⁺ T cells, resulting in decreased infiltration of APCs and activated CD8⁺ T cells, as well as reduced β-2 M and MHC-I expression [333, 334]. LINK-A’s prognostic role may be attributed to its impact on the degradation of TPSN, TAP1, TAP2, and CALR proteins of the peptide-loading complex (PLC), affecting the loading and editing of MHC-I. Consequently, LINK-A inhibitors could enhance the effect of ICIs by increasing the infiltration of hyperactivated CD8⁺ T cells at the tumor level [333]. In DLBCL, MALAT1 upregulates the expression of PD-L1 through miR-195, promoting migration and immune escape mediated by CD8⁺ T cells. Inhibiting MALAT1 could reverse this effect [335]. However, additional clinical validation is required to establish immunoregulatory ncRNAs as viable therapeutic targets for improved leukemia management.

RNA interference approaches

RNA interference (RNAi), a cellular regulatory mechanism found in most eukaryotes, has been harnessed as a method of drug action in the creation of RNAi-based therapies [336]. This natural process, known to researchers for over two decades, involves short RNA strands, specifically siRNAs, leading to targeted gene silencing [337]. Short, double-stranded RNAs known as siRNAs attach to certain messenger RNA sequences to start a chain reaction that cleaves and degrades the target mRNA, thus blocking the production and function of the target gene [338]. siRNAs offer significant therapeutic potential, providing a means to selectively target and silence the mRNA products of genes, which were previously deemed “undruggable” targets [339]. The majority of protein-coding genes can now be decoded and annotated thanks to the human genome, which facilitates the production of complementary siRNA molecules for subsequent protein silencing. However, because they function at the protein level, conventional small-molecule medications demand more structural accuracy and a more involved development procedure [340, 341]. The journey from theoretical to practical drug-related knowledge in the development of siRNA-based drugs has spanned nearly two decades. In August 2018, two decades after the discovery of RNAi, the FDA approved the first siRNA drug, patisiran [342, 343]. This was followed by the approval of givosiran in November 2019 [344] and lumasiran in November 2020 [345]. At present, seven siRNA drugs, including vutrisiran, nedosiran, inclisiran, fitusiran, teprasiran, cosdosiran, and tivanisiran, are in the late stages of Phase 3 clinical trials, with some nearing FDA approval [346, 347]. The difficulty of site-specific delivery, in which large anionic siRNA molecules must cross physiological barriers to enter the cytoplasm of target cells, has slowed the development of siRNA therapeutics. To overcome the delay in realizing the therapeutic potential of RNA interference, stability and specificity have been improved through the use of chemical modifications and delivery methods [347].

Antibodies that bind to either CTLA-4 or PD-1 thereby relieving immune inhibition, have shown promising clinical results in patients [348]. However, only a small percentage of patients had a lasting response to the therapy. Moreover, a high percentage of patients experienced severe side effects, especially those treated with anti-CTLA-4 therapy compared to anti-PD1 therapy [349]. These effects are likely due to the systemic administration of the antibodies causing polyclonal activation of autoreactive T cells. Therefore, additional targeted strategies to inhibit the expression of these co-inhibitory molecules are needed. In this regard, Hobo et al. utilized RNAi to reduce the expression of PD-1 ligands in Dendritic cells (DCs) [350]. The expansion of MiHA-specific CD8⁺ T cells in mice was also boosted by siRNA silencing of PD-1 ligands on DC vaccines [351]. Recently, a new cationic lipid formulation, referred to as SAINT-18, which is compatible with GMP manufacturing, has been used to deliver PD-L1 and PD-L2 siRNAs to DCs [352, 353]. DCs silenced for PD-L and loaded with mRNA encoding for MiHA demonstrated a greater potential for activating MiHA-specific T-cells than control DCs. The data suggests that the immunogenic function of DCs can be enhanced by silencing PD-1 ligands, leading to stronger antigen-specific CTL responses in vitro models and anti-cancer immunity in various mouse cancer models [354]. SiRNAs elicit potent CTL responses by inhibiting certain inhibitory molecules. Chemically produced siRNAs or shRNAs may be added to DCs at no further expense through electroporation. Chemical alterations and lipid-based siRNA delivery techniques provide an alternative to electroporation. The successful targeting of siRNAs to T cells in the bloodstream implies that they may be developed for use as cancer immunotherapies in clinical settings [348]. Malignancies and autoimmune diseases can result from B-cell malfunction. Inadequate siRNA delivery techniques have impeded the development of RNAi-based therapies. Utilizing αCD38 antibody-LNPs encapsulating CycD1 siRNA, an effective and non-immunogenic method has been devised to suppress CycD1 expression in a mouse model of human Mantle Cell Lymphoma [355]. The study showed that encapsulating siRNA into layer-by-layer nanoparticles coated with a targeting antibody allows for the targeted delivery of siRNA to B cells. Unlike the failed use of siRNA therapies in hematologic malignancies because of cell resistance, this approach provides a viable treatment plan for B-cell malignancies [356]. The LbL-np is a siRNA delivery device that encloses the target gene in polyelectrolyte layers to shield it from bloodstream nucleases. Its outer layer, which is dual-targeting (CD20/CD44), guarantees accurate binding to blood cancer cells. The pro-survival protein BCL-2 was silenced both in vivo and in vitro using this method. The dual-targeted nanoparticle's systemic injection caused apoptosis and reduced blood cancer cells' ability to proliferate, indicating that LbL nano assemblies are a potentially effective way to deliver therapeutic siRNA [357].

Conclusions and future perspectives

NcRNAs play a pivotal role in gene regulatory networks, and advancements in genomics and biotechnology have positioned them as promising therapeutic targets in hematological malignancies [15, 30]. Dysregulation of small non-coding RNAs is often linked to altered gene expression in cancer cells [358, 359]. This seminal discovery has paved the way for significant strides in the development of innovative and more potent cancer drugs [360]. lncRNAs operate through different mechanisms compared to miRNAs in cancer, suggesting that their targeting could unveil key tumorigenesis mechanisms [361]. Artificial modulation of ncRNA expression can also restore sensitivity to conventional chemotherapy [362]. Because miRNAs regulate post-transcriptional processes and have dual roles as oncogenes and tumor suppressors in a variety of malignancies, including leukemia and lymphoma, they are the subject of much research in the field of leukemia [313, 363].

In recent times, ncRNAs have been identified as predictive biomarkers for cancer immunotherapy [364]. These biomarkers could offer early evaluation of immunotherapeutic responses, patient prognoses, and cancer recurrence. Various ncRNAs regulate distinct pathways within the cancer microenvironment. However, molecular biomarker indicators are still in their infancy, and no studies have elucidated technical constraints associated with using ncRNA biomarkers for monitoring immunotherapy response [365]. Further research is needed to ensure the reliability of clinical applications. The combination of ncRNA biomarker analysis could reveal novel immunomodulatory agents and therapeutic targets, bolstering the clinical application of cancer immunotherapy [366, 367]. The concurrent use of ncRNAs to modulate specific tumor cell signaling pathways and immunotherapeutic interventions could enhance treatment responses and improve patient prognoses. The high concentration of miRNA in liquid biopsies and tumor tissue may be the reason for the restricted screening of short and lncRNAs as biomarkers in the circulatory systems of cancer patients [113].

The precision of ncRNA-based treatments could be enhanced by directing efforts toward the precursors of ncRNAs. This would involve the use of nucleic acid oligonucleotides or peptides to inhibit their synthesis and maturation [33]. Despite promising results from pre-clinical studies and initial clinical trials, the effective clinical application of ncRNA-based immunotherapy remains unrealized [368]. However, as our understanding of the role of ncRNA in tumor immunity and immunotherapy expands, there is optimism that ncRNA-based treatments could emerge as innovative cancer therapies [20]. Advanced genomic techniques are employed to identify functionally relevant miRNA-mRNA target pairs that regulate the growth of leukemia, which will likely be advantageous for pre-clinical models [369, 370]. The need for better miRNA delivery vehicles for successful therapy is highlighted by the ability of miRNA sponges and anti-miRNA oligonucleotides to silence aberrant miRNAs and the ability of nanoparticle vectors to target oncogenic lncRNAs through the effective delivery of small interfering RNAs [371]. It is possible to modify high-affinity antisense oligonucleotides to decrease carcinogenic lncRNAs via degradation, altering RNA and protein interactions, or alternative splicing. By enabling steady transfection of RNA products into tissues, lentiviral vectors efficiently introduce the siRNA sequence into the intended cell type [372].

The interaction between ncRNAs and immune checkpoints is intricate. The interplay between lncRNAs and immune checkpoints contributes to cancer progression. The ncRNA types play specific roles in advancing the research of tumor resistance and the creation of new drug targets or immunotherapy alternatives. However, the precise roles of lncRNAs on immune checkpoints are not fully understood and necessitate further investigation. The targeted influence of ncRNAs on immune checkpoints suggests potential advancements in leukemia immunotherapy. Notwithstanding their wide range of uses in cancer treatment, ncRNAs in leukemia pose some obstacles to their therapeutic application, such as the intricate cellular milieu and the requirement for a safe and effective delivery mechanism. Furthermore, innovative strategies must be developed to mitigate RNA degradation and thereby increase their bioavailability. While the field of ncRNAs is extensively researched, the exploration of their role as biomarkers and therapeutic targets in leukemia is still in progress.

Author contributions

Samira Anvari: Methodology, Investigation, Visualization, Writing-Original draft preparation, Data Curation, Software, Visualization, Resources, Formal analysis, Writing- Reviewing and Editing; Mohsen Nikbakht: Software, Writing- Reviewing and Editing; Mohammad Vaezi: Software, Writing- Reviewing and Editing; Sedigheh Amini Kafiabad: Funding acquisition, Conceptualization, Project, Supervision; Mohammad Ahmadvand: Funding acquisition, Conceptualization, Project, Supervision.

Availability of data and materials

By ordering from the corresponding author.

Declarations

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Not Applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.